The present invention relates generally to synthesizing clones of Norwalk virus and to making probes to Norwalk and related viruses. It also relates to methods of detection and characterization of Norwalk and related viruses.
Norwalk virus is one of the most important viral pathogens causing acute gastroenteritis, the second most common illness in the United States (Dingle et al., 1953; Kapikian and Chanock, 1985). Up to 42% of cases of viral gastroenteritis have been estimated to be caused by Norwalk or Norwalk-like viruses (Kaplan et al., 1982). Both water and foodborne transmission of Norwalk virus has been documented, and particularly large epidemic outbreaks of illness have occurred following consumption of contaminated shellfish including clams, cockles, and oysters (Murphy et al., 1979; Gunn et al., 1982; Wilson et al., 1982; Gill et al., 1983; DuPont 1986; Morse et al., 1986; Sekine et al., 1989). An increase in fish and shellfish-related food poisonings has recently been noted and attributed to increased recognition of these entities by clinicians as well as to increased consumption of seafood (Eastaugh and Shepherd, 1989). Norwalk virus was discovered in 1973. However, knowledge about the virus has remained limited because it has failed to grow in cell cultures and no suitable animal models have been found for virus cultivation. Human stool samples obtained from outbreaks and from human volunteer studies, therefore, are the only source of the virus. Still the concentration of the virus in stool is usually so low that virus detection with routine electron microscopy is not possible (Dolin et al., 1972; Kapikian et al., 1972; Thornhill et al., 1975). Current methods of Norwalk virus detection include immune electron microscopy and other immunologic methods such as radio immunoassays (RIAs) or a biotin-avidin enzyme linked immunoabsorbent assays (ELISAs) which utilize acute and convalescent phase serum from humans. To date, no hyperimmune serum from animals has been successfully prepared due either to insufficient quantities or unusual properties of the viral antigen. Preliminary biophysical characterization of virions has indicated particles contain one polypeptide (Greenberg et al., 1981), but efforts to characterize the viral genome have failed. Therefore, these viruses have remained unclassified.
1. Dingle J, Badger G, Feller A et al. 1953. A study of illness in a group of Cleveland families: 1. Plan of study and certain general observations. Am. J. Hyg. 58:16-30.
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3. Dolin R, Blacklow N R, DuPont H, Formal S, Buscho R F, Kasel J A, Chames R P, Hornick R, and Chanock R M. 1971. Transmission of acute infectious nonbacterial gastroenteritis to volunteers by oral administration of stool filtrates. J. Infect. Dis. 123:307-312.
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13. Kapikian A Z, Wyatt R G, Dolin R, Thornhill T S, Kalica A R, and Chanock R M. 1972. Visualization by immune electron microscopy of a 27-nm particle associated with acute infectious nonbacterial gastroenteritis. J. Virol. 10:1075-1081.
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15. Morse D L, Guzewich J J, Hanrahan J P, Stricof R, Shayegani M, Deibel R, Grabau J C, Nowak N A, Herrmann J E, Cukor G, and Blacklow N R. 1986. Widespread outbreaks of clam and oyster-associated gastroenteritis: role of Norwalk virus. New Engl. J. Med. 314:678-681.
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It is therefore an object of the invention to characterize the Norwalk and related virus genomes by synthesizing and cloning a cDNA library.
It is an associated object of the invention to deduce amino acid sequences from the cDNA.
Another object of the invention is to develop a method of preparing polyclonal and monoclonal antibodies to the Norwalk and related viruses.
Still another object of the invention is to develop a method of making probes to detect Norwalk and related viruses.
A further object of the invention is to use the cDNA or fragments or derivatives thereof in assays to detect Norwalk and related viruses in samples suspected of containing the viruses.
A still further object of the invention is to express proteins to measure antibody responses.
A nucleotide sequence of the genome sense strand of the Norwalk virus cDNA clone according to the presently preferred embodiment of the invention intended to accomplish the foregoing objects includes the nucleotide sequence shown in Table 1. Within the nucleotide sequence are regions which encode proteins. The nucleotide sequence of the Norwalk virus genome, its fragments and derivatives are used to make diagnostic products and vaccines.
Other and still further objects, features and advantages of the present invention will be apparent from the following description of a presently preferred embodiment of the invention.